Spatholobus parviflorus seed lectin (SPL) is a heterotetrameric lectin, with two α and two β monomers. In the crystal structure of SPL α monomer, two residues at positions 240 and 241 are missing. This region was modeled based on the positional and sequence similarities. The role of metal ions in SPL structure was analyzed by 10 ns molecular dynamics simulation. MD simulations were performed in the presence and absence of metal ions to explain the loss of haemagglutinating property of the lectin due to demetallization. Demetallized structure was found to deviate drastically at the metal binding loop region. Affinity of different sugars like N-acetyl galactosamine (GalNAc), D-galactose and lactose towards the native and demetallized protein was calculated by molecular docking studies. It was found that the sugar binding site got severely distorted in demetallized lectin. Consequently, sugar binding ability of lectin might be decreasing in the demetallized condition. Isothermal titration calorimetric (ITC) analysis of the sugars in the presence of native and demetallized protein confirmed the in silico results. It was observed after molecular dynamics simulations, that significant structural deviations were not caused in the quaternary structure of demetallized lectin. It was confirmed that the structural changes modified the sugar binding ability, as well as sugar specificity of the present lectin. The role of metal ions in sugar binding is described based on the in silico studies and ITC analysis. A comprehensive analysis of the ITC data suggests that the sugar specificity of the metal bound lectin and the loss of sugar specificity due to metal chelation are not linear.
17 independent crystal structures of family I uracil-DNA glycosylase from Mycobacterium tuberculosis (MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5' position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.
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